Biophysical perspectives to understanding cancer-associated fibroblasts

The understanding of cancer has evolved significantly, with the tumor microenvironment (TME) now recognized as a critical factor influencing the onset and progression of the disease. This broader perspective challenges the traditional view that cancer is primarily caused by mutations, instead emphasizing the dynamic interaction between different cell types and physicochemical factors within the TME. Among these factors, cancer-associated fibroblasts (CAFs) command attention for their profound influence on tumor behavior and patient prognoses. Despite their recognized importance, the biophysical and mechanical interactions of CAFs within the TME remain elusive. This review examines the distinctive physical characteristics of CAFs, their morphological attributes, and mechanical interactions within the TME. We discuss the impact of mechanotransduction on CAF function and highlight how these cells communicate mechanically with neighboring cancer cells, thereby shaping the path of tumor development and progression. By concentrating on the biomechanical regulation of CAFs, this review aims to deepen our understanding of their role in the TME and to illuminate new biomechanical-based therapeutic strategies.


I. INTRODUCTION
The tumor microenvironment (TME) is established as a pivotal element in cancer progression within the annals of oncology.Its importance was first noted by Paget in 1889 with the observation of selective metastatic behavior influenced by the surrounding stroma. 1 This initial observation was elaborated upon by Van den Hooff, who explored stromal engagement in malignancy, highlighting the dynamic interactions within the stromal microenvironment. 2 Significant advancements in our understanding of TME were made by Sonnenschein and Soto in "The Society of Cells," where they emphasized the importance of cellular interplay within the TME, laying the groundwork for the Tissue Organization Field Theory (TOFT). 3Subsequent research by Park et al. delved into the TME's regulatory impact on malignant phenotypes, reinforcing its integral role in the initiation and progression of cancer. 4Intercellular and matrix communication has been fundamental in understanding tumor biology.][7] Fibroblasts, integral to maintaining tissue homeostasis and extracellular matrix (ECM) production, undergo a malignant transformation within the TME to become cancer-associated fibroblasts (CAFs).[10] The diversity of CAFs is striking, with distinct subpopulations exhibiting various morphologies, functions, and roles in cancer development.The integration of advanced biochemical and single-cell "omics" tools has broadened our understanding of CAFs. 11However, this approach, while insightful, offers a limited perspective of the full spectrum of CAF functions within the TME.Recent bioengineering and biophysical advances reveal that physical forces, in conjunction with chemical signals, are instrumental in dictating cell behavior and intercellular communication.
This review highlights the morphological and mechanical characteristics of CAFs, offering insights into how mechanotransduction and biomechanical sensors intricately contribute to their functional capabilities.Our exploration extends to the dynamic physical interplay between CAFs and stromal cells, elucidating their complex contributions to the orchestration of tumor progression.This discourse not only underscores the significance of the biophysical characteristics of CAFs but also sets the stage for innovative therapeutic interventions in oncology.Additionally, we delved into the potential of biomaterialbased technologies for CAF reprogramming and examined in vitro methodologies that replicate the ECM nano-topography, thereby offering refined control over CAF phenotypes and functionalities.By mapping the intricate details of CAF biophysical properties and interactions, this review aims to lay the foundation for future research directions and clinical strategies.The insights presented here have the potential to revolutionize current cancer treatment paradigms, steering toward therapeutic innovations that leverage the mechanical and structural intricacies of the TME.

II. BIOPHYSICAL CHARACTERISTICS OF CAFS
The biophysical attributes of CAFs are pivotal, given their direct influence on tumor development and progression.Embracing these attributes presents a potential pathway to augment the current method of CAF subtype identification, which relies exclusively on biochemical markers.Each CAF subtype displays unique biophysical traits, such as cellular morphology, motility, contractility, and mechanical properties.These differences often translate into either tumor-promoting or tumor-inhibiting potential. 10We provide a comprehensive overview of CAFs' biophysical traits, categorized into morphology, motility, and mechanical properties.

A. Morphological variability and motile characteristics of CAF subtypes
CAFs' morphology and dynamic traits are inherently tied to their gene expression profile.Hence, these characteristics likely serve as comprehensive indicators of CAF function at the cellular level. 12onsequently, incorporating both morphological and dynamic characteristics with biomolecular markers could provide a more detailed depiction of the diverse CAF subtypes.
Historical research has identified substantial morphological differences between normal fibroblasts and CAFs.It has been observed that CAFs generally have a larger spreading area, 13,14 which is often linked to the myofibroblastic characteristics of specific subgroups within the population. 15,16oving away from the traditional view of CAFs as a homogeneous population, Pelon et al. have classified heterogeneous CAFs based on their biomolecular markers, adopting a more nuanced perspective to studying CAF biology. 17Among the four subtypes of CAFs they identified (CAF-S1 to S4), emphasis was placed on two subsets, which were both myofibroblastic subtypes (CAF-S1 and CAF-S4).Although both subtypes possess myofibroblastic features, distinctive differences in functional characteristics, such as cellular traction forces, were observed.CAF-S1 exhibited a smaller cell spreading area and a higher aspect ratio, while CAF-S4 displayed a broader spreading area and a lower aspect ratio.These observations affirm that CAF morphology is a vital biophysical determinant of CAF behavior.
For instance, a study by Cao et al. suggested that the spreading area of CAFs can determine their subtype and function, potentially allowing them to transition between myofibroblastic and inflammatory states through modifications by hydrogel network density. 18This study emphasizes the complex nature of CAFs, whose morphological features are just one aspect, necessitating a comprehensive, holistic approach to effectively differentiate CAFs from normal fibroblasts.
0][21][22][23][24][25] Notably, this contractility feature is most prominently observed in a selected CAF subset named myCAFs, a myofibroblast-like phenotype, 18,[26][27][28][29][30][31] which aligns with the primary functions of CAFs and involves remodeling the ECM through mechanical forces.This contractile behavior of CAFs is governed by a complex interplay of signaling pathways, including Rho-ROCKmyosin, JAK1, and the influence of cytokines like oncostatin M, TGF-b, Calponin 1, as well as the transcription factor YAP. 25 Additionally, caveolin 1 32 and a-SMA are implicated in this process, 20,33 although the exact relationship between a-SMA and contractility remains an ongoing subject of investigation.The degree of a-SMA expression has been extensively investigated and found to correlate consistently with the strength of fibroblast spreading, contractility, and focal adhesion formations in various situations. 34,35-SMA, an actin isoform, serves as the definitive marker for identifying activated fibroblasts, smooth muscle cells, and blood vessels.35 It is a standard biomarker for identifying CAFs in the stromal environment and is also a shared marker for myofibroblasts due to their acquired contractile potential.8,36,37 The activated state of fibroblasts is distinguished by the presence of stress fibers containing a-SMA, contributing to increased contractility and altering the morphology of activated fibroblasts.CAFs rich in a-SMA expression display a distinctive morphology characterized by oriented stress fibers, augmented lamellipodia, and a concurrent alteration in inherent stiffness.Although a-SMA is associated with cell contraction, it is unclear if a-SMA actively drives contraction or is a passive by-product, as evidenced by studies suggesting TGF-b-induced contraction may occur independently of a-SMA.[41][42][43][44][45] Moreover, CAFs also acquire different motile behaviors compared to normal fibroblasts.For instance, in gastric CAFs, Ishimoto et al. observed an overexpression of RHBDF2, leading to enhanced cell motility.46 Similar to morphological diversity, CAFs' motility also reflects their heterogeneous nature.Costea et al. reported that two distinct subtypes of CAFs in oral squamous cell carcinoma (OSCC) manifested varied motile characteristics and different influences on cancer cells. 47 Notaly, CAF-N (similar transcriptomic profile of normal oral fibroblasts) displayed enhanced migratory tendencies compared to both CAF-D (more divergent transcriptomic profile of normal oral fibroblasts) and normal oral fibroblasts.Collectively, these studies illuminate the potential of integrating morphological and motile features of CAFs with biomolecular markers to refine our understanding of CAF subtypes.

B. Mechanical properties of CAFs
The mechanical properties of CAFs are as heterogeneous as their morphological traits, which arise from their diverse cytoskeletal arrangements.In an effort to understand these differences, the study by Stylianou et al. employed atomic force microscopy (AFM) to assess Young's modulus of pancreatic CAFs and normal fibroblasts, finding that myofibroblastic pancreatic CAFs are less stiff than their normal counterparts on a 3 mg/ml collagen substrate ($14 kPa). 48On a softer substrate with a lower collagen concentration substrate ($4 kPa, 0.5 mg/ml), however, the myofibroblastic pancreatic CAFs and normal fibroblasts exhibited comparable Young's moduli.The differential mechanical sensitivity refers to how each cell type's elastic modulus changes in response to the stiffness of the underlying substrate. 48ormal fibroblasts appear to adjust their cellular elasticity more significantly in response to substrate stiffness, whereas myofibroblastic pancreatic CAFs do not exhibit the same degree of sensitivity.This finding aligns with observations that normal fibroblasts develop more aligned stress fibers on rigid substrates, whereas CAFs maintain their stress fiber alignment independent of substrate stiffness.These results may collectively suggest that CAFs, despite varied substrate conditions, retain a consistent biomechanical signature, which could be reflective of their pathological role in the TME.
Further complexities in characterizing CAF biomechanics were noted in the work of Jaeschke et al., demonstrating that prostate CAFs exhibit a higher Young's modulus than normal prostate fibroblasts, with these variations being influenced by factors, such as ECM density and measurement methodologies. 49Such differences highlight the need for careful consideration of experimental design when investigating the biomechanical properties of CAFs.
The diversity in biomechanical behaviors observed among CAF subtypes suggests influences from their origin or activation state.To interpret the interconnectedness of morphological and mechanical properties and their collective impact on cancer progression, integrating morphological, motility, mechanical assessments, and transcriptomic analysis is imperative.This comprehensive approach not only aids in understanding the complex interplay between CAF characteristics but also serves as a foundation for identifying and developing therapeutic strategies targeting the biomechanical properties of CAFs to suppress cancer progression.

III. BIOPHYSICAL INTERACTIONS OF CAFS IN TME
Cellular interactions and communication are pivotal in sculpting the TME. 50Beyond the inherent biophysical characteristics of CAFs, their interactions with ECM and other neighboring cells are instrumental in TME dynamics.Such interactions shape the TME and significantly influence CAF phenotypes and behaviors.Given that CAFs respond to a multitude of physical cues, alterations in CAF-ECM dynamics, cell-to-cell interactions, and other biophysical elements like ECM stiffness, microarchitecture, compressive stress, and traction forces can modulate CAF activity [Fig.1(a)].In this review, we discuss the biophysical interactions of CAFs within the TME, with a specific emphasis on ECM remodeling and cell-cell interactions.

A. CAF-ECM interactions
2][53][54][55] By contracting their actin cytoskeleton, CAFs generate heightened traction forces, restructuring the matrix and enhancing stiffness. 56The reciprocal biophysical interplay between CAFs and the ECM influences CAF behaviors; for example, CAFs respond to integrin-mediated mechanical cues while applying forces that aid ECM transformation. 57,58able I presents a comprehensive overview of CAFs and their critical functions within the TME, highlighting the interaction with the ECM mechanical properties of cancer tissue across various organs, including the lung, liver, breast, bone, and prostate.
Alterations in ECM stiffness initiate mechanosignaling pathways that activate CAFs, fostering a dynamic and active stroma contributing to tumor progression. 24,57,96,97Moreover, CAFs exert mechanical forces on the ECM, modifying the orientation and organization of ECM fibers. 27,52,980][101][102] CAFs establish a self-reinforcing loop by maintaining activation through a persistent contractile phenotype, transforming the ECM into a highly reactive desmoplastic stroma. 96,100,103Continuous contraction of CAFs and adopting a contractile form generate mechanical forces via integrins.These forces increase the stiffness of the ECM and activate latent TGF-b1, a crucial player in fibrotic remodeling and stromal activation.This activation triggers the deposition of collagen and fibronectin, causing a rise in ECM rigidity and shifts in its composition.The extensive remodeling and breakdown of the ECM affect interactions between cells and the ECM, altering the abundance and composition of fibronectin and collagen.Consequently, this process reduces the spacing between ECM networks and ligands, impacting cell-ECM interactions.Integrins play a bidirectional role in coordinating this mechanical modification of the ECM.
Mechanotransduction involves integrins sensing ECM stiffness through mechanisms, such as the recruitment of Talin1 and Kindlin2 by Discoidin Domain Receptor 2 (DDR2), leading to collagen-binding integrin activation. 57Additionally, av integrins facilitate stiffness perception and cellular response through focal adhesion kinase, triggering processes like AMPKa activation and autophagy, supporting adjacent cancer cell growth. 109A comprehensive overview of the myriad integrins and their roles in mechanotransduction within CAFs is listed in Table II.
In force generation and matrix remodeling, a5b1 integrin enhances cancer cell invasion via ECM remodeling, while a11b1 integrin, upregulated in desmoplastic tumors, promotes cancer cell invasiveness through force-dependent contractility and collagen remodeling. 23,28,83,110,111av integrins, especially avb3, influence fibrosis, directional migration, and cancer cell invasion by mediating contractility, fibrillary adhesion formation, and fibronectin fibrillogenesis. 23,1145][116][117][118][119] A list of integrins involved in CAF force generation can be found in Table I.Integrin-mediated force generation is key in matrix stiffening, a hallmark feature of aggressive tumors, where integrins convey cellular contraction forces to the matrix, aiding in its organization and enhancing cancer cell migration. 52,112,120Additionally, CAF-secreted enzymes, like lysyl oxidases (LOXs), contribute to matrix cross-linking and stiffening, further contributing to desmoplasia by cross-linking collagen fibers. 121,122uture investigations should focus on the diversity of integrinmediated force generation in mechanosensing pathways and its effect on gene expression and CAF subtype functions.Employing biophysical techniques and single-cell analysis will elucidate the heterogeneity and its implications on stromal biomechanics.

Fluid flow and CAF phenotype
4][125] Interstitial fluid flow (IFF), though characterized by low velocity, exerts a substantial influence on CAF activity.It triggers myofibroblast differentiation, ECM remodeling, and stiffening, attributed mainly to TGF-b signaling. 126,127Fluid shear stress, comparable to the mechanical forces exerted by solid stress, fosters the development of a denser and more intricate matrix network.While hindering fluid transport, this network paradoxically enhances cell motility, likely due to the creation of differential shear gradients across cells. 127ecent studies employing 3D cultures and microfluidic models have shed light on how IFF influences CAF behavior, [128][129][130][131][132] revealing its role in promoting Endothelial to Mesenchymal Transition (EndMT) during CAF differentiation.Constant unidirectional IFF not only drives fibroblast migration toward cancer cells but also correlates with increased expression of CAF markers, such as MMP-9, MMP-14, FAP, vimentin, and a-SMA. 131Moreover, IFF-induced shear stress  activates normal fibroblasts into CAFs through the IGF-PI3K signaling axis, emphasizing the fluid flow's influence on CAF function and differentiation. 133AFs are not mere bystanders to IFF within the TME but also active regulators.The integrin a11b1, expressed by CAFs for instance, is noted for its regulatory capacity on interstitial fluid pressure, influencing the intensity of IFF across various cancers, possibly by influencing the collagen network. 134Such dynamic flow can distort chemokine distribution, enabling the guided migration of cancer cells and CAFs, thereby supporting invasive behavior and metastatic potential.Additionally, the presence of CAFs alongside circulating tumor cells (CTCs) in the bloodstream invites speculation on their possible role in modulating IFF, potentially impacting tumor cell behavior in metastatic niches. 135,136n essence, CAFs are both shaped by and shaping the fluidic landscape of the TME.Their reciprocal relationship with IFF commands further exploration to fully grasp the influence of fluid mechanics on the nuanced roles of CAF subtypes in the evolution and dissemination of cancer.

B. Mechanical communication between CAF and stromal cells
Historically, the emphasis on cell-to-cell communication has centered on chemical signaling.However, newer research underscores the significance of physical interactions in these communications. 21,118,137,138e dynamic interplay between junctional proteins (JPs), including adherens junctions, gap junctions, tight junctions, and nanotunnels, between CAFs and cancer cells is emerging as a critical determinant in cellular recognition and stromal function regulation [Fig.1(c)], Table III.

Adherent junctions of CAFs
Adherens junctions (AJs) primarily facilitate the transmission of intercellular forces via direct cell-to-cell contact, forming mechanical links between the cytoskeletons of adjacent cells. 146Within CAFs, cadherins like N-cadherin and OB-cadherin are present and linked with fibroblast differentiation and high contractile activity. 141ifferent cell types express cadherins, such as E-, N-, and Pcadherin, each playing a distinct role in transmitting intercellular forces among stromal cells.The upregulation of N-cadherin in myofibroblasts, including CAFs, promotes directional migration and filopodia formation while also facilitating mechanical linkage with cancer cells or other stromal cells, thereby enhancing their ability to migrate and invade. 139Filopodia and lamellipodial-like structures enable direct mechanical communication between CAFs and stromal cells, as observed in two-dimensional cultures. 147Notably, CAFs and cancer cells can form heterotypic adhesions between E-cadherin and Ncadherin, promoting the transfer of intercellular forces and furthering cancer cell invasion. 140ancer cells can suppress p53 in surrounding fibroblasts, leading to the development of CAFs and promoting cancer invasion through direct cell-to-cell contact.This suppression of p53 may involve TSPAN12-mediated b-catenin signaling within the fibroblasts.Additionally, TSPAN12 upregulates the secretion of the CXC chemokine ligand 6 (CXCL6), which effectively promote invasion.These findings suggest that TSPAN12 and CXCL6 are potential therapeutic targets to disrupt CAF-mediated lung cancer progression. 148Direct physical interactions between CAFs and cancer cells, or other stromal cells, significantly regulate CAF and cancer cell phenotypes and functions. 43pon activation, fibroblasts exhibit mechanical coupling through AJs, enabling the transmission of local contractile events to adjacent activated myofibroblasts.This contraction creates a mechanical feedback loop that coordinates and recruits connected cells, promoting tissue remodeling and providing information on the contractile status of neighboring cells. 149,150During the transformation of fibroblasts into myofibroblasts, the composition of changes in AJs, with OB-cadherin replacing N-cadherin.This switch allows OB-cadherin to bind with a-SMA, reinforcing the mechanical stability of the myofibroblast population. 141This switching of cadherins may also play a role in the interactions between CAFs and other stromal or cancer cells.The expression of nectin-1, a Ca2 þ -independent cellular adhesion molecule, in CAFs has been correlated with heightened invasion, metastasis, and reduced overall survival in PDAC patients. 151This highlights the potential involvement of CAFs in modulating cell-cell adhesion strength through intercellular contact composition and differential cadherin expression ratios.

Tight junctions (TJs) in CAFs
Exploring the presence of TJs within stromal populations in desmoplastic lesions and their potential role in supporting barrier functions in cancer therapies offers intriguing research opportunities.In a study by Karagiannis et al., occludin, a TJ protein, was discovered in CAF cohorts within desmoplastic lesions of colorectal cancer patients, suggesting that fibroblasts may undergo a context-dependent switch to express claudin-11, potentially contributing to a more collective migratory behavior. 142This intriguing discovery raises the possibility that CAFs harness TJ's machinery to adopt a collective configuration and migrate together as cohorts, potentially influencing tumor behavior. 22oreover, the crucial role of TJ strands in fibroblasts, acting as a fence-like structure, cannot be overlooked as they establish a physical barrier between the apical and basolateral membranes, contributing to the maintenance of barrier integrity. 152As cancer therapies often aim to breach these barriers, understanding TJs within stromal subsets could pave the way for innovative strategies that target tumor expansion by dismantling these protective barriers.

Gap junctions and tunneling nanotubes in CAFs
Gap junctions (GJs), facilitated by connexins, pose challenges in overcoming chemotherapy resistance within the TME, forming physical barriers between CAFs and cancer cells that compromise treatment effectiveness. 143,144Decoding their role in mechanical interactions and metabolic shifts between CAFs and cancer cells is crucial for addressing chemo-resistance. 146n the realm of intercellular communication, tunneling nanotubes (TNTs) have emerged as pivotal conduits, connecting distant cells and facilitating the exchange of genetic material. 145,153While CAFs predominantly provide resources to fuel cancer cells, cancer cells exploit TNTs to influence CAF behavior. 145Deciphering the mechanisms behind TNT formation elucidates their profound involvement in promoting or inhibiting tumor growth.However, their fragile nature renders TNTs vulnerable to mechanical disruptions, potentially compromising their intercellular connections.5][156] Moreover, these TNTs may relay mechanical signals, affecting cell migration and function.An in-depth understanding of the mechanotransduction processes linked to TNTs in CAF-cancer cell interactions offers insight into their dual role in promoting and inhibiting tumor progression.
Effective communication is the cornerstone of the dynamic interplay between CAFs and their stromal counterparts.AJs, TJs, GJs, and

C. Physical interactions of CAFs with inflammatory cells
Although the physical interaction between CAFs and inflammatory cells is often overshadowed by biochemical pathways, it has recently been revealed to be a vital regulator of tumor dynamics.Tsoumakidou's comprehensive review describes major subtypes of CAFs, such as myCAFs, iCAFs, and apCAFs. 157The review highlights their impact on the infiltration of immune cells, emphasizing the need for additional research to explore their specific interactions.
MyCAFs play a role in the formation of a dense ECM network, which acts as a physical barrier that restricts the entry of immune cells, specifically the effector T cells that are crucial for combating tumor growth.][160][161] Specifically, ECM proteins secreted by CAFs, such as big-h3, create obstacles for T-cell infiltration by interacting with integrin b3 on CD8þ T cells.This interaction hinders the immune response against tumors. 162However, a promising approach to overcome this CAFmediated barrier involves the use of T cells armed with membraneanchored and tumor-targeted IL-12, particularly attIL12 cells.These engineered T cells likely achieve this by stimulating the production of IFNc upon interaction with tumor cells.IFNc, in turn, suppresses growth factors secreted by CAFs, leading to CAF apoptosis and a remodeled tumor microenvironment.This remodeled environment allows for better infiltration of effector T cells, overcoming the barrier previously established by the ECM and CAFs. 163he phenotypic adaptability of myCAFs has been highlighted in recent investigations.In response to the dense ECM, myCAFs have been observed to undergo morphological transformations, adopting a more rounded shape and shifting toward a high IL-6 secreting profile akin to iCAFs within three-dimensional physio-mimetic culture systems. 18This plasticity introduces additional complexity to the myCAF/iCAF-ECM-T cell interface, suggesting that myCAFs, after contributing to ECM densification, may transform into iCAFs, with implications for T-cell interaction and suppressed anti-tumor activity.iCAFs typically express a cytokine-rich, ECM-deficient profile, suggesting a possible plateau in ECM production following their transition from myCAFs, which could further complicate immune cell infiltration.The differing adhesive molecule expressions between iCAFs and myCAFs could affect their physical engagement with T cells, altering T cell activation, mobility, and efficacy within the TME.Tsoumakidou also posits that myCAF and iCAF phenotypes might not be distinct entities but rather extremes on a continuum of fibroblastic differentiation.
Furthermore, Tuveson and colleagues have unveiled antigenpresenting CAFs (apCAFs) bearing MHC class II in pancreatic cancer. 164These apCAFs, when studied ex vivo, may lose MHC II expression and take on myCAF-like characteristics, underscoring the necessity of investigating apCAFs within their native tumor context for their role in sustaining effector CD4þ T cell responses. 157,164,165uture studies should dissect the functional distinctions between classical antigen-presenting cells (APCs) and apCAFs, with a specific focus on the adhesion mechanisms governing their interaction with T cells.Physical interaction between CAFs and T cells is illustrated in Fig. 2.

IV. THERAPEUTIC STRATEGIES TARGETING BIOPHYSICAL PROPERTIES OF CAFS A. Current approaches to targeting CAFs in cancer therapy
7][168][169] Therapeutic targeting focuses on mechanisms triggering CAF activation and associated signaling pathways. 121Numerous signaling cascades significantly impact the biological activities of CAFs and the interplay between CAFs and cancer cells.For example, both the canonical and noncanonical TGF-b signaling pathways in CAFs hold potential as targets, especially concerning their crosstalk with tumor cells.Similarly, the PI3K/AKT/mTOR, Wnt, MAPK, and JAK/STAT pathways are intensively targeted by simultaneously suppressing the crosstalk of CAF with cancer cells.Numerous reviews have already detailed the therapeutic targeting of CAFmediated signaling pathways with a current list of clinical trials. 10,26,121dditionally, innovative strategies, such as CAF depletion, inhibition, infiltration disruption, reprogramming, and the development of CAFdirected therapeutics, vaccines, and immune-based technologies, are under investigation to counteract CAF-mediated tumor progression.The TME and downstream effectors of CAFs are under intensive investigation for potential new treatments. 166espite these advances, the versatile nature of CAFs, which can shift between different subtypes, presents a challenge in enhancing tumor control.Deep insights into the balance between tumorpromoting and restricting CAFs and their dynamic interactions in the TME are essential as they uncover potential targets.CAFs experience significant biophysical shifts, adopting a tumor-promoting phenotype characterized by morphological changes, enhanced migration, and increased force generation.Consequently, strategies focusing on the biophysical regulation of CAFs are promising in cancer therapy.This section provides a detailed look into methods for altering the biophysical properties of CAFs, harnessing their pro-tumorigenic potential, and unlocking their anti-tumor capabilities.

B. Potential of targeting biophysical cues to regulate CAFs
Reducing contractility and mechanosensing in CAFs contribute to the disruption of the mechanical feedback loop, resulting in reduced desmoplastic reactions and ECM remodeling, thereby suppressing cancer invasion. 170In a study conducted by Chronopoulos et al., all-trans retinoic acid (ATRA), an active metabolite of vitamin A, restores mechanical quiescence in pancreatic stellate cells (PSCs) by downregulating actomyosin contractility mediated by the retinoic acid receptor beta (RAR-b). 171ATRA treatment in three-dimensional (3D) organotypic models diminishes the ability of CAFs to generate high traction forces, adapt to mechanical cues from the surrounding environment, and inhibit force-mediated ECM remodeling.ATRA has now emerged as a promising stromal targeting agent in combination with chemotherapy for PDAC. 172nhibiting focal adhesion kinase (FAK) activity and targeting proteins involved in cellular contractility, such as myosin II or Rhoassociated protein kinase (ROCK), helps deter force generation in CAFs. 170When combined with chemotherapy, FAK inhibitors induce a significant decrease in metastasis in PDAC by reducing CAF force generation and ECM remodeling. 173Similarly, inhibiting ROCK, a downstream effector of the Rho GTPase that regulates cell contractility, disrupts the contractile properties of CAFs and impacts their functional interactions with cancer cells.ROCK inhibitors hold great promise as therapeutic targets due to their involvement in stromal remodeling, stiffness sensing, contraction, and the formation of stress fibers in CAFs. 169Phase I trials investigating the use of ROCK and FAK inhibitors have highlighted the importance of targeting contractility and force-mediated ECM remodeling.
Targeting key signaling pathways involved in mechanotransduction, such as the YAP/TAZ pathway or the Hippo pathway, holds the potential to disrupt the activation of CAFs and mitigate their protumorigenic effects. 174,175Activated YAP in CAFs, known for promoting malignancy and angiogenesis, contributes to increased fibrosis surrounding tumor cells.The mechanoactivation of YAP/TAZ in CAFs facilitates feed-forward interactions and impedes the dynamic interplay between CAFs and other cells within the tumor ecosystem. 25,176Systematically inhibiting YAP/TAZ in CAFs emerges as a potentially effective treatment strategy to prevent metastasis and overcome chemoresistance. 175nhibiting enzymes involved in ECM remodeling, such as matrix metalloproteinase (MMPs) and LOXs, have shown potential. 26,171While preclinical models have demonstrated promising results with MMP inhibitors in preventing CAF-mediated ECM stiffening and desmoplasia, 177,178 LOX2 inhibitors have also yielded encouraging results in PDAC trials. 179However, LOX2 inhibitors have shown limited improvements in patient survival.This discrepancy could be attributed to the multifaceted role of CAFs and the necessity for precisely identifying CAF phenotypic variations associated with LOX2 secretion.
Recognizing that these inhibitors predominantly target CAFs characterized by high contraction and force generation is crucial, neglecting the importance of addressing CAF subtypes lacking these physical traits.Therefore, it is imperative to understand CAFs comprehensively based on their phenotypical signatures, including their physical characteristics.Simplistic pharmacological normalization might not suffice to address challenges within the TME.
In our comprehensive review of the biophysical regulation of CAFs, we emphasize the immense potential of modulating biophysical cues to alter CAF behavior.Furthermore, we explore ex vivo biomaterial-based platforms capable of precisely regulating the biophysical traits of CAFs.

C. Biomaterials in understanding and modulating CAF function
Extensive studies have focused on harnessing the potential of biomaterials to regulate cell fate, both in vitro and in vivo. 180These materials offer a unique opportunity to manipulate both the biochemical and biophysical properties, influencing crucial cellular processes, such as morphology, migration, and differentiation potential, as illustrated in Fig. 3.We highlight specific mechanical properties, including biomaterial stiffness, ECM network density, ligand density, and ligand patterning to steer the phenotypic behavior of CAFs ex vivo.
Emerging studies utilizing the collagen and alginateinterpenetrating network (CoAl-IPN) hydrogel system have provided significant insight into the mechano-mediated regulation of CAF behavior and their interaction with tumor cells.For instance, in breast CAFs, a compliant hydrogel network facilitates spreading and augments pro-tumorigenic activities by activating YAP/TAZ mechanosignaling pathways and upregulating CAF-specific genes.In contrast, a stiffer and more cross-linked hydrogel network can constrain the spreading and phenotype of breast CAFs (b-CAFs), subsequently diminishing their pro-tumorigenic paracrine functions. 18,181 related investigation on colorectal CAFs demonstrated that variations in the network density of the CoAl-IPN can lead to a reversible phenotypic shift between inflammatory and myofibroblastic states.This shift is governed by the polymer network's confinement effect and the ROS-HIF1-a mechanotransduction-signaling axis.The myofibroblastic state prompts epithelial-mesenchymal transition (EMT) in colorectal adenocarcinoma cells, aiding their dissemination and potentially accelerating tumor progression, whereas the inflammatory state is associated with anti-tumorigenic effects and improved patient survival outcomes in colorectal cancer.18 The inflammatory state, indicated by rounded cell morphology, reduced alpha-SMA levels, and elevated IL-6 levels, may be prompted by physical constraints within the tumor stroma.This environmental interaction can induce a transition from pro-tumorigenic, spindleshaped myCAFs to rounded, potentially tumor-suppressive iCAFs, underlining the significance of physical cues in influencing CAF phenotypes toward tumor suppression.
However, the therapeutic manipulation of CAF morphology and biomechanical properties requires a complex understanding of the distinct physical attributes associated with their tumor-promoting and suppressing functions.It is crucial to identify these characteristics precisely to prevent inadvertently diminishing the beneficial actions of certain CAF subtypes.Ongoing and future research should focus on delineating the specific physical features of CAFs along their functional continuum and understanding how these aspects relate to tumor dynamics.Achieving this will be crucial for developing targeted therapeutic strategies that judiciously modulate CAF functions, supporting their tumor-suppressive capacity while preserving their advantageous roles within the tumor microenvironment.
In a related study conducted by our group, we found that fibrous architecture with high network density in gelatin fibers confines normal fibroblasts, preventing their activation into CAFs by limiting cell spreading and altering morphology. 182These findings collectively emphasize FIG. 3. Engineered platforms for modeling and measuring CAFs.A multifaceted approach that includes biomaterial strategies, advanced cellular models, and mechanical characterization tools to model and measure the distinct behavior of CAFs to uncover novel therapies.(Schematic was created using BioRender Software.) the critical role of 3D network density and spatial cellular confinement as key biophysical determinants that profoundly influence CAF phenotypic plasticity and activation.Modulation of cell-ECM binding interactions regulates a spectrum of cellular functions, including motility, differentiation, growth, and morphological responses.Traditional analyses of cell-matrix dynamics often overlook the inherent viscoelastic properties of tissues, which are crucial for accurately recapitulating tissue mechanics across various pathological states, as elucidated by Chaudhuri et al. 183 Our focus encompasses the role of such properties in CAF behavior, particularly within the ECM's interstitial niches.
Recent work by Lin et al. utilizing a biomimetic hydrogel system designed to emulate the stiffness and viscoelastic properties of pancreatic cancer tissue revealed that increased matrix stiffness, irrespective of viscoelastic changes, correlates with a reduction in activated CAF markers. 184The study further noted that dynamic stiffening of the matrix curtailed CAF spreading and surface area, implying a limitation in their migratory and contractile abilities.Although there were observable shifts in cell cycle-associated gene expression, CAF proliferation remained notably unchanged. 185This suggests that replicating dynamic stiffening observed in tumor microenvironments could modulate CAF activity, downregulating activation markers and confining cellular expansion.
Additionally, an earlier study by the same research team emphasized the influence of ECM composition, observing that increased stiffness in conjunction with hyaluronic acid buildup fostered a more elongated CAF morphology, possibly due to enzymatic expression changes. 186This finding underscores the intricate relationship between matrix viscoelastic properties and CAF function.By mimicking the native ECM and manipulating its mechanical attributes, biomaterials present promising avenues for mitigating the pro-tumorigenic functions of CAFs, offering potential for innovative cancer therapies.
CAFs form fibrillary adhesions while remodeling fibronectin (FN).Mechanical cues highly regulate the assembly of these adhesions.The contractile forces produced by CAFs can unfold FN, creating tension and revealing cryptic binding sites.FN remodeling, influenced by tension, changes ligand binding site arrangements and overall ECM organization. 187While the nanoarchitecture of the ECM is well established on biomaterial platforms, understanding the nanoscale distributions of ligand binding sites that are crucial for stromal cell behavior, particularly CAFs, remains limited.Researchers have crafted tailored ligand presentation platforms at varying scales and spacing to unravel the impact of the spatiotemporal organization of ligands. 188,189This approach, extensively studied in stem cells and cancer cells for differentiation and chemoresistance functions, offers a unique opportunity for understanding CAF behavior.As force-generating cells, modulating ligand density at different scales can reshape CAF physical traits and dedifferentiate their phenotype at the cell-ECM axis.These platforms hold significant promise for engineering CAFs, aiming to restrain tumor progression ex vivo.
Building upon the earlier discussion highlighting CAF morphological features as crucial markers for their subtypes, the profound impact of ECM nano-topography on cell morphology presents an enticing opportunity.Utilizing nanopatterned substrates for precise control over CAF morphology and potential reprogramming to their original lineages emerges as an exciting strategy.[192][193] While nanopatterning precisely controls cell shape, its indirect influence on cytoskeletal arrangements governing contractility is a challenge.A recent study led by Cabezas et al. highlights that nanoscale anisotropic features within a patterned matrix exert direct control over actin fibers within confined shapes, offering a programmable approach within defined geometries. 190Moreover, mechanical reprogramming of fibroblasts through laterally confined micropatterned substrates induces a shift toward stem-cell-like phenotypes, reducing DNA damage and enhancing cytoskeletal gene expression and actomyosin contractility. 194Overall, utilizing nanopatterned substrates combined with biomaterials that mimic the cellular niche offers a powerful platform for the precise manipulation of the geometrical characteristics of CAFs.This advanced approach holds promise for directing the reversion of CAFs to their original phenotypes.
Incorporating the CAF study within the context of biomaterial environments paves the way for therapeutic innovations.By exploring the capacity of biomaterials to influence CAF phenotypes and functions, we can pinpoint novel therapeutic targets and craft innovative strategies.By Bridging foundational research with clinical possibilities, this integration transforms insights gained from in vitro models into actionable therapeutic interventions.The potential to develop treatments that specifically alter CAF activity, thereby hindering tumor growth and enhancing patient prognosis, is an exciting frontier in cancer therapy.

D. Bridging the gap between biophysical and molecular markers
CAFs exhibit remarkable heterogeneity, yet they share several molecular markers that confer distinct biophysical characteristics, such as morphology and motility, influencing tumor behavior.Key markers include a-SMA, FSP-1/S100-A4, FAP, tenascin-C, PDGFR-a/b, CD90, MMPs, MHC-II, and podoplanin (PDPN), which are indicative of the CAFs' role within the TME. 8,10 a-SMA: A model for understanding CAF phenotypes a-SMA is a prominent CAF marker, closely associated with enhanced fibroblast contractility and ECM remodeling.This reflects CAFs' contractile phenotype, as previously discussed regarding their morphological variability and motility characteristics.
The spatial distribution of a-SMA significantly affects fibroblast functions and contractility.A recent study by Kwartler et al. emphasizes that the nuclear accumulation of a-SMA in smooth muscle cells markedly enhances the expression of contractile genes. 195Importantly, a-SMA, when polymerized into stress fibers, not only induces contractility but also offers valuable insights into the evaluation of its localized position and correlation with morphological features.These biophysical traits are particularly significant in a-SMA-positive CAFs (a-SMA þ CAFs), also known as myCAFs, which are crucial in ECM remodeling and force generation.
Furthermore, a study led by Barbazan et al. reveals that a-SMAexpressing CAFs, characterized by high contractility, actively compress cancer cells by forming an actomyosin ring. 192This compression is achieved through a coordinated contraction within the a-SMA expressing CAF layer.This study highlights the multifaceted role of a-SMA in orchestrating both structural and functional changes in CAFs.

FSP-1 and mechanotransduction
We have reexamined the significance of the DDR2 gene in CAF mechanotransduction.In a study using FSP-1 cre mice, deletion of the DDR2 gene in FSP-1-expressing CAFs led to altered extracellular matrix (ECM) architecture and reduced tumor stiffness.It impeded lung metastasis, indicating that FSP-1 positive CAFs influence ECM mechanics and metastatic outcomes through the DDR2 pathway. 57outhern et al. further expounded on the role of FSP-1(S100A4) in myofibroblast trans-differentiation and fibrosis, where FSP-1 facilitates the reorganization of actomyosin, contributing to a contractile phenotype. 196The subtleties of FSP-1 positive CAFs necessitate further detailed investigation to elucidate their roles in mechanotransduction.This process highlights the importance of FSP-1 in the mechanical and functional evolution of CAFs.Nonetheless, it emphasizes the need for comprehensive research into FSP-1 positive CAFs, which could reveal further insights into their mechano-effector activities or their developmental lineage within the CAF population.

FAP and ECM remodeling
Lee et al. highlighted the role of FAP-positive CAFs in ECM remodeling, which orchestrates the alignment of collagen fibers to foster an environment conducive to cancer invasion. 197The study elucidates the mechanotransduction function of FAP in cancer progression, highlighting how the FAP-positive CAF-derived matrix can enhance cancer cell invasion engaging b1-integrin/FAK.

E. Challenges and future directions in developing biophysical-based therapies for CAFs
Developing biophysical properties-based therapies for CAFs poses significant challenges.One main obstacle is the variability within CAFs; they show different behaviors and have varied roles in the TME.Precisely targeting CAF subpopulations with unique biophysical traits may unlock effective therapeutic interventions.This would involve studying the mechanical, flow-related, and shape-related properties of CAFs using tools like magnetic tweezers, rheometers, and AFM-based methods (Fig. 3).
To better understand the roles and types of CAFs, we should consider both biochemical and biophysical information (Fig. 3).Future directions in this field involve identifying novel biophysical targets and devising innovative strategies to modulate CAF behavior.This could include investigating new techniques like drug delivery inspired by cell mechanics and incorporating biophysical cues into combination therapies.However, successfully implementing these strategies requires an integrative approach, employing a diverse set of mechanobiological tools to unravel the inherent mechanical behavior of CAFs.

V. CONCLUSION AND IMPLICATIONS FOR FUTURE RESEARCH
While the potential of biomolecular markers has been extensively explored, the biophysical attributes of CAFs, such as those impacted by IFF and the associated mechanotransduction within the TME, bring a compelling dimension to our understanding of cancer progression.Integrating both biomolecular and biophysical characteristics affords a more holistic perspective of CAF subtypes and their multifaceted roles within the TME.This integrative approach goes beyond mere geometric measurements, encompassing CAF morphology, motility, mechanical properties, and biomolecular markers.Such an enhanced analytical framework promises to improve the precision in identifying and targeting specific CAF subpopulations therapeutically (Fig. 3).
Exploring the biophysical interactions of CAFs within the TME, particularly their role in CAF-ECM remodeling and intercellular communication, offers novel opportunities for therapeutic strategies.Incorporating insights on the role of fluid dynamics, such as IFF, is instrumental in dictating CAF phenotype and function, revealing a profound influence on ECM remodeling, CAF activation, and subsequent tumor cell interaction and invasion.Therapies targeting CAF-ECM interactions or mechanosignaling pathways have shown promise in reducing CAF activation and restraining tumor progression.In addition, the insights from the physical interactions between CAFs and inflammatory cells could provide novel modalities to modulate immune responses within the TME.These approaches, however, must be carefully optimized to avoid disrupting the intricate balance within the TME.
Innovative approaches, like biomaterial-based platforms, offer precise control over the phenotypic behavior of CAFs by altering the mechanical properties of the ECM and TME.These platforms can potentially shift the balance from pro-tumorigenic to anti-tumor activities.Still, they necessitate a deep understanding of the complex interactions within the TME to ensure efficacy and safety in clinical applications.Yet, these strategies must be developed with an awareness of the delicate balance within the TME and the possibility of unforeseen consequences.
The complexity of the TME and the consequential role of CAFs underline the necessity for an interdisciplinary approach.A collaborative effort that unites diverse fields ranging from oncology and mechanobiology to materials science and bioengineering is essential for constructing a comprehensive understanding of the TME.Such a multidisciplinary strategy is not merely advantageous but essential for unraveling the subtleties of CAF subtypes and exploring innovative therapeutic paths.
Ultimately, a balanced perspective that recognizes both the potential benefits and the inherent risks is critical for advancing cancer treatment.Interdisciplinary collaboration will be key in developing safer, more effective cancer therapies that enhance patient outcomes.By embracing this approach and integrating both biomolecular and biophysical insights, we can progress toward demystifying the nuances of CAF functions and unlocking new avenues for combating cancer.

FIG. 1 .
FIG. 1. Biophysical perspectives to understanding cancer-associated fibroblasts.(a) Impact of physical cues from extracellular matrix (ECM) in regulating phenotype and function of CAFs.(b) The role of integrin-mediated force generation and mechanotransduction across diverse mechanosignaling pathways that influence gene expression and the heterogeneous functions of CAFs.(c) The dynamic interplay between junctional proteins (JPs), including adherens junctions, gap junctions, tight junctions, and nanotunnels, between CAFs and cancer cells is emerging as a critical determinant in cellular recognition and stromal function regulation.(Schematic was created using BioRender Software.)

FIG. 2 .
FIG. 2. Physical interaction between MyCAFs, iCAFs, and ApCAFs with ECM-T cells axis: MyCAFs secrete a dense ECM network that hinders T-cell infiltration.iCAFs exhibit a lower ECM density and potentially altered adhesion interactions with T cells.apCAFs, expressing MHC-II molecules, can present antigens to CD4þ T cells.Arrows indicate the reciprocal communication between these components, influencing T cell movement and function.(Schematic was created using BioRender Software.)

TABLE II .
List of integrins that regulate mechano-transduction and force generation in CAFs.

TABLE III .
List of junctional proteins in CAF-mediated mechanical communication in stroma.TNTs emerge as key players in orchestrating the exchange of essential signals.The altered expression of JPs by CAFs adds complexity to their dual roles as promoters and inhibitors of tumor growth.The interplay of JPs with actin stress fibers intricately shapes CAF morphology during high-stakes, force-driven activities.A potent biomarker can be identified to decode distinct CAF subtypes by unraveling the enigma of JP expression patterns during CAF development.